Novel serine-threonine kinase gene

ABSTRACT

A novel gene having the consensus sequence of a serine-threonine kinase active site has been isolated by the suppression subtractive hybridization method which comprised of preparing a library of genes expressed specifically in fetal livers and isolating clones from this library at random. This gene presumably participates in cell growth control because it is highly expressed, especially in actively growing cells, and exhibits a significant homology with a vaccinia virus B1R kinase gene. Thus, it can be utilized as a target for developing cell growth inhibitors or antitumor agents.

[0001] This application is a continuation-in-part of InternationalApplication PCT/JP97-04855 filed Dec. 25, 1997, which claims priorityfrom Japanese Patent Application No. 8/357864 filed Dec. 27, 1996.

TECHNICAL FIELD

[0002] The present invention relates to a novel serine-threonine kinasegene.

BACKGROUND OF ART

[0003] Fetal tissues are comprised of many undifferentiated cells thatproliferate actively, highly activated cells, nascent vascularendothelial cells, and so on. Although the activity of these cells infetal tissues is stringently regulated and inhibited as individualsmature, the state of fetal tissues can be considered similar to that ofa solid tumor except that the activity is regulated. Therefore, some ofthe genes expressed specifically or more strongly in fetal tissues(fetal genes) can be genes involved in the phenomena characteristic ofsolid tumors such as abnormal growth, immortalization, infiltration,metastasis, and angiogenesis. In addition, some diseases other thantumors are also supposed to arise because fetal genes, which arerepressed in a normal living body, are abnormally activated. Therefore,genes involved in various diseases such as tumors can be screened byisolating and analyzing fetal genes.

[0004] However, there are still few reports on systematic analysisfocusing merely on fetal genes from these viewpoints, and at presentthere is a far from perfect understanding of these gene groups.

DISCLOSURE OF THE INVENTION

[0005] An objective of this invention is to isolate genes expressedspecifically in fetal tissues and to screen genes related to diseases.

[0006] The present inventors thought that fetal tissue cells could be amodel for solid tumor cells and that genes involved in diseases such astumors could be screened by isolating and analyzing fetal genes.Furthermore, the present inventors thought it possible to develop amedicine with a novel action mechanism by designing drugs targeting thegenes. Based on these thoughts, the present inventors have tried toisolate fetal genes.

[0007] Specifically, the present inventors prepared a subtractionlibrary with many genes expressed specifically in fetal livers (or morestrongly than adult livers) by the suppression subtractive hybridizationmethod, extracted clones from this library at random, and analyzed theirstructure. By doing so, the present inventors succeeded in isolating anovel gene, VRK1, having the consensus sequence of a serine-threoninekinase active site. The present inventors also performed a data basesearch based on the amino acid sequence deduced from the isolated gene.The present inventors thus have found this gene product exhibits asignificant homology with B1R kinase, which is presumably involved inDNA replication of vaccinia virus. In addition, the present inventorsfound human EST having a very high homology with this gene in thedatabase and isolated its full-length cDNA, VRK2. Analyzing theexpression of the two isolated genes in various cells by northern blotanalysis showed that these genes are strongly expressed, especially inactively growing cells such as human fetal livers, testes, and varioustumor cell lines. Furthermore, the present inventors have found that theVRK1 protein actually has protein kinase activity.

[0008] Thus, the present invention relates to novel serine-threoninekinase genes, VRK1 and VRK2. More specifically, the present inventionrelate to:

[0009] (1) a protein having the amino acid sequence of SEQ ID NO: 2, ora protein having the same amino acid sequence where one or more aminoacids are added, deleted, or substituted and having serine-threoninekinase activity,

[0010] (2) a protein having the amino acid sequence of SEQ ID NO: 4, ora protein having the same amino acid sequence where one or more aminoacids are added, deleted, or substituted and having serine-threoninekinase activity,

[0011] (3) a protein encoded by a DNA sequence that hybridizes with theDNA sequence of SEQ ID NO: 1 or its complementary sequence and havingserine-threonine kinase activity,

[0012] (4) a protein encoded by a DNA sequence that hybridizes with theDNA sequence of SEQ ID NO: 3 or its complementary sequence and havingserine-threonine kinase activity,

[0013] (5) a DNA encoding the protein of any one of (1) to (4),

[0014] (6) a vector comprising the DNA of (5),

[0015] (7) a transformant carrying the vector of (6),

[0016] (8) a method of producing the protein of any one of (1) to (4),wherein the method comprises cultivating the transformant of (7),

[0017] (9) an antibody binding to the protein of any one of (1) to (4),

[0018] (10) an antisense DNA against the DNA of (5) or part of it,

[0019] (11) a method of screening compounds having inhibitory activityof serine-threonine kinase activity of the protein of any one of (1) to(4), wherein the method is comprised of

[0020] (a) contacting the protein of any one of (1) to (4) with asubstrate to be phosphorylated by this protein in the presence of a testcompound to detect the kinase activity of the protein of any one of (1)to (4), and

[0021] (b) comparing the kinase activity detected in step (a) with thatdetected in the absence of the test compound and selecting a compoundthat lowers the kinase activity of the protein of any one of (1) to (4).

[0022] The present invention relates to novel serine-threonine kinases,“VRK1” and “VRK2.” The nucleotide sequence of the “VRK1” cDNA and theamino acid sequence of the protein are shown in SEQ ID NO: 1 and 2,respectively. In addition, the nucleotide sequence of the “VRK2” cDNAand the amino acid sequence of the protein are shown in SEQ ID NO: 3 and4, respectively. “VRK1” cDNA has a significant homology with B1R kinase,which is presumably involved in DNA replication of vaccinia virus. Thegene is also characterized by its strong expression in actively growingcells such as fetal livers, testes, and various tumor cell lines. Inaddition, overexpression of “VRK1” protein drastically increases thegrowing activity of NIH3T3 cells. These facts imply “VRK1” is involvedin the regulation mechanism of cell growth. “VRK1” protein has proteinkinase activity, which presumably plays an important roll in theregulation of cell growth. “VRK2” has a high homology with “VRK1,”especially in the serine-threonine kinase site. “VRK2,” like “VRK1,” hasa significant homology with B1R kinase, and the gene is characterized byits strong expression in actively growing cells such as fetal livers,testes, and various tumor cell lines. These facts imply “VRK2” has thesame function as that of “VRK1.”

[0023] “VRK1” and “VRK2” proteins can be prepared as recombinantproteins with recombinant DNA techniques or as natural proteins. Therecombinant proteins can be prepared, for example, by cultivating cellstransformed with the DNAs encoding these proteins, as will be describedlater. Natural proteins can be isolated from fetal livers, testes, ortumor cell strains such as HeLa S3, in which these proteins are highlyexpressed, by a method well-known to one skilled in the art, such asaffinity chromatography with the antibodies of the present invention asdescribed later. Either polyclonal or monoclonal antibodies can be used.The polyclonal antibodies can be prepared from, for example, serum fromsmall animals such as rabbits immunized with these proteins by, forexample, ammonium sulfate precipitation, protein A- or protein G-columnchromatography, DEAE ion exchange chromatography, affinitychromatography using a column coupled with these proteins, etc. Themonoclonal antibodies can be prepared as follows. First, a small animalsuch as a mouse is immunized with these proteins. The spleen isextracted from the mouse and dissociated to cells. The resulting cellsare fused to mouse myeloma cells using a reagent such as polyethyleneglycol, and the clone that produces antibodies against these proteins isscreened from the fusion cells (hybridoma) thus generated. The hybridomathus obtained is then transplanted into a mouse abdominal cavity.Ascites is collected from the mouse and purified by, for example,ammonium sulfate precipitation, protein A- or protein G-columnchromatography, DEAE ion exchange chromatography, affinitychromatography using a column coupled with “VRK1” or “VRK2” protein,etc. If the antibodies obtained are to be used for administering to ahuman body (for antibody therapy or the like, etc.), humanizedantibodies or human antibodies should be used to decreaseimmunogenicity. An example of methods for humanizing antibodies is theCDR graft method, in which an antibody gene is cloned from monoclonalantibody-producing cells and its antigenic determinant is transplantedto an existing human antibody. Besides, human antibodies can be directlyprepared just like usual monoclonal antibodies by immunizing a mousewhose immune system is replaced with a human immune system.

[0024] Furthermore, one skilled in the art can prepare not only natural“VRK1” and “VRK2” proteins (SEQ ID NO: 2 and 4, respectively) but alsoproteins with substantially the same function as that of the naturalproteins, if needed, by replacing amino acids in the proteins by awell-known method. Besides, mutations of amino acids in proteins canoccur naturally. Thus, mutant proteins with serine-threonine kinaseactivity that are generated by introducing amino acid substitution,deletion, or addition into the natural proteins are also included in theproteins of the present invention. Methods for amino acid alteration,for example, a site-directed mutagenesis system using PCR (GIBCO-BRL,Gaithersburg, Maryland), the oligonucleotide-mediated site-directedmutagenesis method (Kramer, W. and Fritz, H J (1987) Methods inEnzymol., 154:350-367), and the Kunkel method (Methods Enzymol. 85,2763-2766 (1988)), are well-known to one skilled in the art.Furthermore, usually ten or less, preferably six or less, and morepreferably three or less amino acids are substituted. For example,proteins functionally equivalent to the VRK1 or VRK2 protein can beproduced by conservative amino acid substitutions at one or more aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, s leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). The site where substitution, deletion, or addition isintroduced is not particularly limited as long as the serine-threoninekinase activity is maintained. From the viewpoint of protein activity,the addition, deletion, or substitution of amino acids should beperformed in a region other than the region corresponding to theconsensus sequence of a serine-threonine kinase active site and to theconsensus sequence of a protein kinase ATP binding site. Moreover,serine-threonine kinase activity of a protein can be detected, forexample, by the method described in Example 9, mentioned later.

[0025] Furthermore, one skilled in the art can usually isolate DNAshaving a high homology with the DNA encoding “VRK1” or “VRK2” protein(SEQ ID NO: 1 or 3, respectively) based on the DNA or the part of itusing a hybridization technique (Sambrook, J. et al., Molecular Cloning2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) and obtainproteins having substantially the same function as VRK1 or VRK2 protein(SEQ ID NO: 2 or 4, respectively) from the DNA. Thus, proteins withserine-threonine kinase activity that are encoded by DNAs hybridizingwith DNA encoding “VRK1” or “VRK2” protein are also included in theproteins of the present invention. Hybridizing DNAs are isolated fromother organisms including, for example, mice, rats, rabbits, andbovines, and so on . Tissues such as fetal livers and testes areespecially suitable for isolating. Thus isolated DNAs encoding proteinshaving substantially the same function as that of “VRK1” or “VRK2”proteins usually have a high homology with the DNA (SEQ ID NO: 1 or 3)encoding “VRK1” or “VRK2” protein, respectively. The term “highhomology” used herein means at least 40% or more, preferably 60% ormore, and more preferably 80% or more of sequence identity at the aminoacid level. From the viewpoint of the protein activity, a high homologyshould be found in the regions corresponding to the consensus sequenceof a serine-threonine kinase active site and to the consensus sequenceof a protein kinase ATP binding site.

[0026] To determine the percent homology of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence) . The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

[0027] To determine percent homology between two sequences, thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3to obtain amino acid sequences homologous to a VRK1 or VRK2 proteinmolecules. To obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used. See http://www.ncbi.nlm.nih.gov.

[0028] Furthermore, the present invention relates to a DNA thatspecifically hybridizes under moderate or highly stringent conditions toa DNA encoding a protein of the present invention and comprises at least15 nucleotide residues. The DNA can be used, for example, as a probe todetect or isolate a DNA encoding a protein of the present invention, oras a primer for PCR amplification. An example is DNA consisting of atIeast 15 nucleotides complementary to the nucleoctide sequence of SEQ IDNO: 2 or NO: 3.

[0029] Standard hybridization conditions (e.g., moderate or highlystringent conditions) are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6, hereby incorporated by reference. Moderatehybridization conditions are defined as equivalent to hybridization in2× sodium chloride/sodium citrate (SSC) at 30° C., followed by one ormore washes in 1×SSC, 0.1% SDS at 50-60° C. Highly stringent conditionsare defined as equivalent to hybridization in 6× sodium chloride/sodiumcitrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 50-65° C.

[0030] Examples of conditions for hybridization to isolate these DNAsare as follows. After prehybridization at 55° C. for 30 minutes orlonger, hybridization is performed by adding labeled probes andincubating at 37° C. to 55° C. for an hour or longer using “ExpressHybHybridization Solution” (CLONTECH). After that, the resulting hybridizedproducts are washed three times for 20 minutes each at room temperaturein 2×SSC and 0.1% SDS then once at 37° C. in 1×SSC and 0.1% SDS. Morepreferably, after prehybridization at 60° C. for 30 minutes or longer,hybridization is performed by adding labeled probes and incubating at60° C. for an hour or longer using “ExpressHyb Hybridization Solution”(CLONTECH). Thereafter, the hybridized products are washed three timesfor 20 minutes each at room temperature in 2×SSC and 0.1% SDS then twiceat 50° C. in 1×SSC and 0.1% SDS. Still more preferably, afterprehybridization at 60° C. for 30 minutes or longer, hybridization isperformed by adding labeled probes and incubating at 68° C. for an houror longer using “ExpressHyb Hybridization Solution” (CLONTECH).Thereafter, the hybridized product is are washed three times for 20minutes each at room temperature in 2×SSC and 0.1% SDS then twice at 50°C. in 0.1×SSC and 0.1% SDS.

[0031] The present invention also relates to the DNAs encoding theabove-described proteins of the present invention. The DNAs of thepresent invention include cDNAs, genomic DNAs, and synthetic DNAs aslong as they encode the proteins of the present invention. The DNAs ofthe present invention can be used to produce the recombinant proteins.Specifically, the recombinant proteins can be prepared by inserting theDNA (for example, the DNA of SEQ ID NO: 1 or 3) of the present inventioninto a suitable expression vector, cultivating the transformant obtainedby introducing the vector into suitable cells, and purifying theexpressed proteins. For example, mammalian cells such as COS, CHO, orNIH3T3 cells; insect cells such as Sf9 cells; yeast cells; and E. colicells can be used for producing the recombinant proteins. Vectors forexpressing recombinant proteins in these cells vary depending on thehost cells. For example, pcDNA3 (Invitrogen) or pEF-BOS (Nucleic Acids.Res. 1990, 18(17), p5322) is used for mammalian cells; “BAC-to-BACbaculovirus expression system” (GIBCO BRL), for insect cells; “PichiaExpression Kit” (Invitrogen), for yeast cells; and pGEX-5X-1 (Pharmacia)or “QIAexpress system” (Qiagen), for E. coli cells. Vectors can beintroduced into host cells by, for example, the method using calciumphosphate, DEAE dextran, or cationic liposome DOTAP (BoehringerMannheim); electroporation; the calcium chloride method; etc. Therecombinant proteins can be purified from the obtained transformants bythe usual methods such as the method described in “The Qiaexpressionisthandbook, Qiagen, Hilden, Germany.”

[0032] Furthermore, the DNAs of the present invention can be used forgene therapy of diseases caused by mutations in genomic DNAs. In genetherapy, the DNAs of the present invention are administered to a livingbody inserted into adenovirus vectors (e.g., pAdexLcw), retrovirusvectors (e.g., pZIPneo) and so on. They can be administered by either exvivo methods or in vivo methods.

[0033] Furthermore, since the proteins of the present invention arepresumably involved in the regulation of cell growth, antisense DNAsagainst the DNAs of the present invention or part of them can be used asinhibitors for developing cell growth or as antitumor agents. Theantisense DNAs are administered to a living body directly or in the formof the vectors into which they have been inserted. The antisense DNAscan be synthesized by methods well known to one skilled in the art.

[0034] The present invention also relates to a method of screeningcompounds having inhibitory activity of serine-threonine kinase activityof the proteins of the present invention. This screening method consistsof two steps. First, the protein of the present invention is caused tocontact a substrate to be phosphorylated by this protein in the presenceof a test compound to detect the kinase activity of the protein of thepresent invention. Second, the kinase activity detected in step (a) iscompared with that detected in the absence of the test compound, and acompound that lowers the kinase activity of the protein of the presentinvention is selected.

[0035] Test compounds used for this screening method are notparticularly limited and are generally low-molecular-weight compounds,proteins (including the above-described antibodies of the presentinvention), peptides, etc. Test compounds are either artificiallysynthesized or natural. Substrates are, for example, casein, IkBαprotein, etc. The kinase activity of the protein of the presentinvention can be detected, for example, by adding ATP havingradioactively labeled phosphate to the reaction system containing theprotein of the present invention and the substrate and measuring theradioactivity of the phosphate attached to the substrate. Specifically,the activity is detected by the method described in Example 9. Thecompounds thus isolated can be used as cell growth inhibitors orantitumor agents. Moreover, the present inventors learned that “VRK1”protein phosphorylates IkBα protein. IkBα is thought to be rapidlydegraded when phosphorylated, thereby releasing and activating NF-kBbound thereto. In addition, NF-kB is well known as a centraltranscriptional regulator that causes widespread immune reactions andinflammation reactions. Therefore, compounds that inhibit the kinaseactivity of the proteins of the present invention can be used asantiphlogistics and immunosuppressants.

BRIEF DESCRIPTION OF THE DRAWING

[0036]FIG. 1 shows the adapters used for constructing the subtractionlibrary.

[0037]FIG. 2A shows the consensus sequence of the active site ofserine-threonine kinase. FIG. 2B shows the consensus sequence of the ATPbinding site of protein kinase.

[0038]FIG. 3 shows the nucleotide sequence of the clone fls223 and itsdeduced amino acid sequences.

[0039]FIG. 4 shows electrophoretic patterns demonstrating the result ofRT-PCR analysis performed to detect expressions of VRK1 and VRK2 genesin fetal and adult livers. In the figure, “A” and “F” represent Adultliver, and Fetal liver. “Low,” “Middle,” and “High” represent the levelof PCR cycles.

[0040]FIG. 5 compares amino acid sequences of VRK1 and B1R.

[0041]FIG. 6 compares amino acid sequences of VRK1 and VRK2.

[0042]FIG. 7 compares amino acid sequences of VRK2 and B1R.

[0043]FIG. 8 shows electrophoretic patterns demonstrating the result ofnorthern blot analysis of the expression of VRK1 and VRK2 genes invarious cells.

[0044]FIG. 9 shows an electrophoretic pattern demonstrating the resultof western blot analysis using anti c-Myc antibody. Cell extracts fromCOS7 cells transfected with plasmid DNA, pcDNA3 (lane 1) orpcDNA3/VRK1myc (lane 2), were examined.

[0045]FIG. 10 shows an electrophoretic pattern demonstrating the resultof northern blot analysis using the VRK1 cDNA as a probe. Total RNAsamples prepared from NIH3T3 cells transfected with plasmid DNA, pCOS(lane 1) or pCOS/VRK1w (lane 2), and from a human hepatoma cell line,HepG2 cells (lane 3), were examined.

[0046]FIG. 11 presents microscopic photographs showing the result ofcolony assay. A pool of NIH3T3 cells transfected with plasmid DNA, pCOS(“pCOS”) or pCOS/VRK1w (“pCOS/VRK1w”) was examined.

[0047]FIG. 12 shows an electrophoretic pattern of purified GST fusionproteins (CBB staining). Fusion proteins with wild VRK1 protein (lane 1)or with a mutant one (lane 2) were examined.

[0048]FIG. 13 shows electrophoretic patterns demonstrating the result ofkinase assay. Added proteins are indicated by “+” on the upper portion.Arrows indicate phosphorylated GST-VRK1 (“A,” autophosphorylation),phosphorylated casein (“C”), phosphorylated GST-IkBα (“I”), andphosphorylated IkBα C-terminal peptide (“P”).

[0049]FIG. 14 shows an electrophoretic pattern demonstrating the resultof kinase assay. Reactions were performed in the presence of variousdivalent cations at various concentrations as indicated on the right.Arrows indicate phosphorylated GST-VRK1 (“A,” autophosphorylation), andphosphorylated casein (“C”).

[0050]FIG. 15 shows an electrophoretic pattern demonstrating the resultof western blot analysis with an antibody against a VRK1 peptide usingK562 cell extracts.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention is illustrated below in detail withreference to examples, but is not to be construed as being limitedthereto.

EXAMPLE 1

[0052] Construction of a Subtraction Library

[0053] A subtraction library was prepared using the PCR-Select™ cDNASubtraction kit (CLONTECH) basically according to the method describedby Luda Diatchenko et al. (Proc. Natl. Acad. Sci. USA, Vol.93,6025-6030, 1996).

[0054] First, double-stranded cDNAs were synthesized from polyA⁺ RNAprepared from human fetal and adult livers by the standard method usingMMLV reverse transcriptase. Next, the respective cDNAs were blunt-endedwith T4 DNA polymerase, then cleaved by RsaI. A part of the cDNAoriginating from fetal liver (tester) was split in two; one of which wasligated with the adapter-1 and the other with the adapter-2 (FIG. 1).Each aliquot was mixed with an excess amount of the adult liver cDNA(driver), denatured by heat, and subjected to the first hybridization at68° C. for 8 hours. Aliquots were then combined without heatdenaturation, mixed further with an excess amount of heat-denatureddriver, and subjected to the second hybridization at 68° C. for about 16hours. The mixture was diluted in the dilution buffer, incubated at 75°C. for 7 minutes to remove the shorter strands of adapters, and used asa template for PCR. By performing PCR with primers corresponding to theadapters, “PCR primer-1” (SEQ ID NO: 5) and “PCR primer-2” (SEQ ID NO:6), cDNAs carrying different adapters on their two ends (subtractedcDNAs) were selectively amplified (suppression PCR). To obtain productswith further selectivity, a portion of the amplified products was usedas a template for PCR with primers “Nested PCR primer-1” (SEQ ID NO: 7)and “Nested PCR primer-2” (SEQ ID NO: 8), which locate further inside ofthe primers; “PCR primer-1” (SEQ ID NO: 5); and “PCR primer-2” (SEQ IDNO: 6). The products were purified using the “QIAquick PCR Purificationkit” (QIAGEN), and cloned into the pT7Blue-T vector (Novagen) by the TAcloning method to create a subtraction library.

EXAMPLE 2

[0055] Sequence Analysis

[0056] Plasmid DNA prepared by the alkali SDS method or products ofcolony PCR were used as a template for sequence reaction. Sequencereaction was performed by the cycle-sequencing method using the ABIPRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit With AmplyTaqDNA Polymerase, FS, and the result was analyzed by the ABI 377 DNASequencer.

[0057] Colony PCR was performed as follows. Colonies carryingrecombinant vectors were directly suspended into PCR reaction mixturesthat contain vector primers, “M13 P4-22 primer” (SEQ ID NO: 9) and “M13P5-22 primer” (SEQ ID NO: 10). After PCR reaction, amplified insert DNAwas separated from unreacted primers and nucleotides by gel filtrationor the like, and used as a template for sequencing.

[0058] As a result, the clone fls223 (261 bp) (later renamed “VRK1”) wasfound to be able to encode an amino acid sequence (FIG. 3) that containsthe consensus sequence of the active site of serine-threonine kinase([Leu, Ile, Val, Met, Phe, Tyr, Cys]-Xaa-[His, Tyr]-Xaa-Asp-[Leu, Ile,Val, Met, Phe, Tyr]-Lys-Xaa-Xaa-Asn-[Leu, Ile, Val, Met, Phe, Tyr, Cys,Thr]-[Leu, Ile, Val, Met, Phe, Tyr, Cys, Thr]-[Leu, Ile, Val, Met, Phe,Tyr, Cys, Thr]) (corresponds to amino acids at 173-185 of SEQ ID NO: 2)(FIG. 2A). In addition, no gene registered in the database completelymatches the nucleotide sequence of this clone. Thus, the gene is a novelone.

EXAMPLE 3

[0059] RT-PCR Assay

[0060] Using polyA⁺ RNA extracted from fetal and adult livers,single-stranded cDNAs were synthesized by the standard method withSUPERSCRIPT™ II RNase H⁻ Reverse Transcriptase (GIBCO BRL). Some of thecDNAs were used as a template for RT-PCR analysis of fls223. PCR wasperformed using TaKaRa Taa (TaKaRa) as Taq polymerase by the hot-startmethod, where the reaction was started by adding TaqStart™ Antibody(CLONTECH). Primers “FLS223 S1 primer” (SEQ ID NO: 11) and “FLS223 A1primer” (SEQ ID NO: 12) were used to amplifyfls223.

[0061] The G3PDH (glyceraldehyde 3-phosphate dehydrogenase) gene, whichis a housekeeping gene equally expressed in various tissues and known tobe influenced only slightly by various inducers on its expression, wasused as a control. G3PDH was amplified using the primers “hG3PDH5′primer” (SEQ ID NO: 13) and “hG3PDH3′ primer” (SEQ ID NO: 14). RT-PCRanalysis confirmed that the clone fls223 is strongly expressed in fetalliver, and its expression was also detected in adult liver (FIG. 4). Thefull-length cDNA was then cloned for more detailed analysis of the gene.

EXAMPLE 4

[0062] Cloning by Rapid Amplification of cDNA End (RACE)

[0063] The Marathon™ Ready cDNA (CLONTECH) or cDNA prepared by theMarathon™ cDNA Amplification Kit (CLONTECH) was used as a template for5′ RACE and 3′ RACE (Chenchik A. et al., CLONTECHniques X, 1, 5-8,1995).

[0064] The primers described above, “FLS223 S1 primer” (SEQ ID NO:. 11)and “FLS223 A1 primer” (SEQ ID NO: 12), were used for 5′ RACE and 3′RACE of VRK1fls223. Using a combination of these primers and a primerAP1 (SEQ ID NO: 15), corresponding to the adapter of the template cDNA,tThe reaction was performed with a combination of these primers and aprimer AP1 (SEQ ID NO: 15), corresponding to the adapter of the templatecDNAbasically consisted of a reaction at 94° C. for (2 minutes); fivecycles of reactions at 94° C. for (30 seconds) and at 68° C. (4minutes); and 30 cycles of reactions at 94° C. for (30 seconds), 62° C.for (1 minute), and 72° C. for (3 minutes;) and followed by a reactionat 72° C. for 10 minutes. TaKaRa Ex Taq (TaKaRa) was used for PCR, andthe reaction was started by the hot-start method by adding TaqStart™Antibody (CLONTECH). After reaction, detected bands were recovered usingthe QIAquick Gel Extraction Kit (QIAGEN), and subcloned into thepT7Blue-T vector (Novagen).

[0065] Analysis of the entire nucleotide sequence revealed that thefull-length fls223 cDNA encodes an open reading frame composed of 396amino acids (refer to SEQ ID NO: 1). In the former part of the aminoacid sequence, there exists a consensus sequence of the ATP binding siteof protein kinase ([Leu, Ile, Val]-Gly-Xaa-Gly-Xaa-[Phe, Tyr, Trp, Met,Gly, Ser, Thr, Asn, His]-[Ser, Gly, Ala]-Xaa-[Leu, Ile, Val, Cys, Ala,Thr]-Xaa-Xaa-[Gly, Ser, Thr, Ala, Cys, Leu, Ile, Val, Met, Phe,Tyr]-Xaa(five times or 18 times)-[Leu, Ile, Val, Met, Phe, Tyr, Trp,Cys, Ser, Thr, Ala, Arg]-[Ala, Ile, Val, Pro]-[Leu, Ile, Val, Met, Phe,Ala, Gly, Cys, Lys, Arg]-Lys) (corresponds to the amino acids 43-71described in the SEQ ID NO: 2) (FIG. 2B), and a consensus sequence ofthe active site of serine-threonine kinase, which is also found in theoriginal clone. Thus, the gene product is assumed to be a novelserine-threonine kinase.

[0066] A homology search of the whole database revealed that the geneshows high homology to the B1R gene product of the Vaccinia virus (J.Gen. Virol., 70, 3187-3201, 1989; J. Gen. Virol., 72, 1349-1376, 1991)(FIG. 5). The B1R gene encodes a protein composed of 300 amino acids andis assumed to be a serine-threonine kinase because the gene contains theconsensus sequences analogous to that of the ATP binding site of proteinkinase and of the active site of serine-threonine kinase. Thefull-length tls223 cDNA and the B1R gene showed relatively high homologyover the entire region as well as in the kinase domain (Smallest Sumprobability in Blast search=2.7e−78). Therefore, the gene is named“Vaccinia virus B1R kinase related Kinase 1” (VRK1).

[0067] B1R kinase is an early gene whose expression is observed in earlystages. It appears several hours after vaccinia virus infection and isthen repressed. It has been shown that in a mutant strain containing apoint mutation on the gene, virus replication stops during DNAreplication. Thus, it has been hypothesized that B1R kinase regulatesvirus DNA replication (J. Biol. Chem., 264, 21458-21461, 1989).

[0068] VRK1 also exhibits an obvious homology to B1R kinase in theregion outside of the serine-threonine kinase domain. Thus, VRK1 mayparticipate in the regulation of cellular DNA replication or, morewidely, in cell growth control, as is the case for B1R kinase in virus.This notion is supported by the fact that the VRK1 genes are morestrongly expressed in tissues such as fetal liver and the testis, wherenumerous actively growing cells exist.

[0069] Furthermore, a public clone “human EST-H80169,” which has anextremely high homology to VRK1, was found by searching the data base.Using the primers “RK A2 primer” (SEQ ID NO: 16) and “RK S1 primer” (SEQID NO: 17) for 5′ RACE and 3′ RACE, the full-length cDNA of the gene wascloned as described for VRK1, and the entire nucleotide sequence wasdetermined. As a result, it was found that the gene encodes an openreading frame composed of 508 amino acids (refer to SEQ ID NO: 3), inwhich the consensus sequence of the active site of serine-threoninekinase exists. Thus, this gene may also encode a novel serine-threoninekinase. The amino acid sequence has an extremely high homology to VRK1,especially near the kinase domain (FIG. 6), and a high homology to thevaccinia virus B1R kinase (FIG. 7). These suggest a close relationshipbetween this kinase and B1R kinase. Thus, it was named “Vaccinia virusB1R kinase related Kinase 2” (VRK2).

[0070] RT-PCR confirmed that VRK2 is also expressed more strongly infetal liver than in adult liver (FIG. 4). The primers “RK S2 primer”(SEQ ID NO: 18) and “RK A2 primer” (SEQ ID NO: 16) were used for RT-PCR.

EXAMPLE 5

[0071] Chromosome Mapping

[0072] Chromosome mapping of the VRK1 and VRK2 genes was performed usingthe GENEBRIDGE 4 Radiation Hybrid Panel (Research Genetics, Inc.)(Nature Genetics, 7, 22-28, 1994). DNA on the panel was used as atemplate for PCR. For VRK1, PCR was performed with a combination of theabove primers (“FLS223 S1 primer” (SEQ ID NO: 9) and “FLS223 A1 primer”(SEQ ID NO: 12)) by a reaction at 94° C. for (5 minutes); five cycles ofreactions at 94° C. for (30 seconds) and at 72° C. for (2 minutes); and30 cycles of reactions at 94° C. for (30 seconds) and at 68° C. for (2minutes; and). This was followed by a reaction at 72° C. for 3 minutes.For VRK2, PCR was performed with a combination of primers “VRK2 Aprimer” (SEQ ID NO: 19) and “VRK2 B primer” (SEQ ID NO: 20) by areaction at 94° C. for (3 minutes);, and 30 cycles of reactions at 94°C. for (30 seconds), 60° C. for (1 minute), and 72° C. for (2 minutes;and). This was followed by a reaction at 72° C. for 5 minutes. Theresulting pattern was analyzed on a database on Internet(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl), and maps wereobtained.

[0073] The VRK1 gene was thus mapped to the position between the STSmarkers “D14S265” and “AFM063XE7” on chromosome 14. Similarly, the VRK2gene was mapped to the position between the STS markers “CHLC.GATA23H01”and “D2S357” on chromosome 2.

EXAMPLE 6

[0074] Northern Blot Analysis

[0075] The expressions of VRK1 and VRK2 mRNA in various human normaltissues and tumor cell lines were analyzed by northern blotting (FIG.8).

[0076] The 5′ terminal fragment of the VRK1 cDNA (upstream region of theHindIII site at nucleotide residue 546) or that of the VRK2 cDNA(upstream region of the EcoRI site at position 426) was labeled with[α-³²P]dCTP by the random primer method using Ready-to-Go DNA labelingbeads (Pharmacia), and used as a probe. Hybridization was performed at68° C. in ExpressHyb Hybridization Solution (CLONTECH) using MultipleTissue Northern (MTN) Blot-Human, Human II, Human Fetal II, and HumanCell line (CLONTECH) according to the method recommended by themanufacturer. Final wash was done at 50° C. in 0.1×SSC, 0.1% SDS, andthe image on the filter processed through hybridization was analyzedwith the BAS-2000II bioimaging analyzer (Fuji Photo Film).

[0077] In FIG. 8, tumor cell lines used are malignant melanoma cells“G361,” lung carcinoma cells “A549,” colorectal adenocarcinoma cells“SW480,” Burkitt's lymphoma cells “Raji,” acute lymphoblastic leukemiacells (T cell) “MOLT-4,” chronic myelogenous leukemia cells “K-562,”uterocervical carcinoma cells “HeLaS3,” and promyelocytic leukemia cells“HL60”.

[0078] The results revealed that VRK1 was expressed relatively highly infetal tissues, and extremely highly in fetal liver. VRK1 was expressedweakly in almost all adult tissues, but it was expressed strongly in thetestis and the thymus. In tumor cell lines, a very strong expression ofVRK1 was observed in six out of eight cell lines.

[0079] The expression pattern of VRK2 was basically similar to that ofVRK1; VRK2 was expressed strongly in fetal liver and the testis.Similarly, it was expressed strongly in tumor cell lines. However, VRK2was not expressed in MOLT-4 cells, in contrast to the pattern of VRK1MRNA.

EXAMPLE 7

[0080] Constructing Expression Plasmid DNAs

[0081] CDNA containing the entire coding region of VRK1 or VRK2 wasamplified by PCR using a combination of primers, VRK1 S1 primer (SEQ IDNO: 21) and VRK1 A1 primer (SEQ ID NO: 22), or VRK2 S1 primer (SEQ IDNO: 23) and VRK2 A1 primer (SEQ ID NO: 24) from CDNA synthesized frompolyA⁺ RNA extracted from human fetal liver. The amplified product wascleaved at the NotI site that is attached to the end of the primers andpurified by agarose gel electrophoresis to obtain DNA fragments of thecorrect size. These were subcloned into the PCOS vector, which waspretreated with NotI and dephosphorylated on its ends withalkaline-phosphatase/CIAP (TaKaRa). This vector contains an EF1αpromoter and enables expressing cloned CDNA strongly in a broad range ofmammalian cell lines. By sequencing the thus-obtained subclones, clones(PCOS/VRK1w, PCOS/VRK2w) without mutation such as PCR error wereselected and used for expression as described below and for furtherconstruction of expression plasmid DNA.

[0082] Plasmids for expressing proteins in which the anti c-Myc antibodyepitope sequence (SEQ ID NO: 25) is attached to the C-terminus wereconstructed as follows. Using about 50 nanograms of the plasmid DNA andwith PCOS/VRK1w or PCOS/VRK2w as a template, PCR was performed with acombination of primers. These included VRK1 MYC1 primer (SEQ ID NO: 26)and VRK1 MYC2 primer (SEQ ID NO: 27), or VRK2 MYC1 primer (SEQ ID NO:28) and VRK2 MYC2 primer (SEQ ID NO: 29), and CDNA with the anti c-Mycantibody epitope attached to the C-terminus of the coding sequence wasamplified. KOD DNA polymerase (TOYOBO) was used as the DNA polymerase.The amplified product was cleaved at the BamHI site that is attached tothe end of the primers and purified by agarose gel electrophoresis toobtain DNA fragments of the correct size. These were subcloned into thepcdna3 vector (Invitrogen), which was digested with BamHI anddephosphorylated on its ends with alkaline-phosphatase/CIAP (TaKaRa). Bysequencing the thus-obtained subclones, clones (pcdna3/VRK1myc,pcdna3/VRK2myc) without mutation such as PCR error were selected, andused for later experiments.

[0083] Expression plasmid DNAs for glutathione-S-transferase (GST)fusion proteins in E. coli were constructed as follows. Using theplasmid DNA PCOS/VRK1w or PCOS/VRK2w as a template, the coding regionwas amplified by PCR with a combination of primers, VRK1 H3 primer (SEQID NO: 30) and VRK1 H4 primer (SEQ ID NO: 31), or VRK2 H3 primer (SEQ IDNO: 32) and VRK2 H4 primer (SEQ ID NO: 33). The amplified product wascleaved at the BamHI site that is attached to the end of the primerspurified by agarose gel electrophoresis to obtain DNA fragments of thecorrect size. These were then subcloned into the PGEX-5X-1 vector(Pharmacia), which was digested with BamHI and dephosphorylated on itsends with alkaline-phosphatase/CIAP (TaKaRa) . By sequencing thethus-obtained subclones, clones (PGEX/VRK1w, PGEX/VRK2w) withoutmutation such as PCR error were selected and used for later experiments.

[0084] A clone with a mutation introduced to the predicted ATP bindingsite within the kinase catalytic domain (Lys at position 71 in the aminoacid sequence of SEQ ID NO: 2 is replaced by Trp) was constructed usingthe Chameleon™ Double-Stranded Site-Directed Mutagenesis Kit(STRATAGENE) as follows. About one microgram of the PGEX/VRK1w plasmidDNA was mixed with primers VRK1 KW1 primer (SEQ ID NO: 34) and aselection primer, Select1 primer (SEQ ID NO: 35), and heat denatured byboiling for 5 minutes. Plasmid DNA and both primers containing mutationwere then annealed by incubating at room temperature for 30 minutes.Next, new DNA strands were synthesized from primers by adding substratenucleotides, DNA polymerase, etc. These were treated with PstI to digestwild plasmid DNA, and introduced into XLmutS competent cells. Afterovernight liquid culture, plasmid DNA was extracted then treated withPstI to digest contaminating wild plasmid DNA. It was then reintroducedinto the competent cells. By sequencing several single isolatedcolonies, a clone (PGEX/VRK1K71W) with an introduced mutation wasselected.

EXAMPLE 8

[0085] Expression in Mammalian Cell Lines

[0086] About 10 micrograms of plasmid DNA, pcdna3/VRK1myc or pcdna3, wasintroduced (transfected) into COS7 cells using SuperFect (QIAGEN).Specifically, about 10⁶ COS7 cells were plated in a 10-cm dish, andcultured overnight. A mixture of 10 micrograms of plasmid DNA and 60microliters of SuperFect was then added to the culture, and the culturewas incubated for about 3 hours. Thereafter, the culture medium wasreplaced with fresh medium. The cells were then cultured for two moredays and collected by detaching in a trypsin-EDTA solution. Cells werewashed once in PBS, disrupted in RIPA buffer (1% NP-40, 10 Mm Tris-Hcl(Ph 7.2), 0.1% sodium deoxychorate, 0.1% SDS, 0.15 M sodium chloride, 1Mm EDTA, 10 micrograms/ml aprotinin, 1 Mm PMSF), and cell extracts wereobtained by centrifugation. The cell extracts were separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected towestern blot analysis using anti c-Myc antibody (SANTA CRUZ). A band ofabout 50 kDa was specifically observed in cells transfected with thepcdna3/VRK1myc plasmid DNA, indicating that VRK1myc protein is expressed(FIG. 9).

[0087] Next, about 7.5 micrograms of plasmid DNA, PCOS/VRK1w or PCOS,was transfected into NIH3T3 cells by the method using cationicphospholipid DOTAP (Boehringer Mannheim). After transfection,transformants were selected by adding G418 (GIBCO-BRL) to the culturemedium to a final concentration of 500 micrograms/ml. Total RNA wasprepared from each pool of transformants by the method using ISOGEN(Wako Junyaku). The total RNA was then subjected to northern blotanalysis using the VRK1 CDNA as a probe. The results confirmed that VRK1MRNA was expressed in a pool of cells obtained by transfection with thePCOS/VRK1w plasmid DNA (FIG. 10). These pools of cells were examined forthe ability to form colonies on soft agar (colony assay). To this end,2×10⁴ cells were suspended in Dulbecco's Modified Eagle Medium (DMEM)containing 10% fetal bovine serum and 0.4% thawed SeaPlaque agarose(TaKaRa), and overlaid on a bottom agarose, which was made of 0.53%SeaPlaque agarose, 10% fetal bovine serum, and DMEM. After two-weekculturing, the pool of cells obtained by transfection with thePCOS/VRK1W plasmid DNA formed a number of colonies that were obviouslylarger than those formed in the pool of cells obtained by transfectionwith the Pcos plasmid DNA. This suggests that overexpression of VRK1confers abnormal growth activities on cells (FIG. 11).

EXAMPLE 9

[0088] Expression of VRK1 Protein in E. coli and Kinase Assay

[0089] Both wild VRK1 protein and mutant VRK1 protein were expressed inE. coli as a fusion protein with GST protein and purified. The E. coliDH5α strain cells carrying the above-described plasmid DNA, PGEX/VRK1w,or PGEX/VRK1K71W were cultured overnight at 37° C. in 10 ml 2×YT medium.Some of the culture was diluted 100-fold with fresh 2×YT medium andcultured at 37° C. until the OD value at 600 nm reached 0.6. IPTG(isopropyl-β-D(−)-thiogalactopyranoside) was then added to the cultureto a final concentration of 0.1 Mm, and the culture was incubatedfurther for several hours. The E. coli cells were collected bycentrifugation, resuspended in PBS containing 1% Triton X-100 and 1%Tween 20, and subsequently disrupted by sonication to solubilizeproteins. From the solubilized samples, wild VRK1 protein and mutantVRK1, which were expressed as a fusion protein with GST, were purifiedby affinity chromatography using glutathione Sepharose4B (Pharmacia).These proteins were subjected to SDS-PAGE and stained with CoomassieBrilliant Blue (CBB) to confirm their purity (FIG. 12). GST protein andGST-IkBα protein were prepared in the same manner.

[0090] Kinase assay was performed on a total of 50 microliters of areaction mixture containing 0.2 micrograms of wild or mutant VRK1protein, 50 Mm Tris-Hcl (Ph 7.2), 1 Mm dithiothreitol (DTT), 2 Mm or 10Mm of divalent cation (Mg, Mn, Zn, Ca), a maximum of 5 micrograms ofsubstrate protein, and 1 microliter of [γ-³²P]-ATP (3000 Ci/Mm, 10mCi/ml [Amersham]). In some experiments, another buffer systemcontaining 40 Mm Hepes (Ph 7.4), 1 Mm DTT, and 2.5 Mm EGTA was used.

[0091] Specifically, the reaction was first performed in the presence of10 Mm Mg at 37° C. for 30 minutes using, as a protein substrate, histone(Nakalai), casein (Sigma), myelin basic protein/MBP (Sigma), GST,GST-IkBα, or IkBα C-terminal peptide (SEQ ID NO: 36). The reactionmixture was subjected to SDS-PAGE, and the radioactivity ofphosphorylated proteins was analyzed with a BAS2000II bioimaginganalyzer (Fuji Photo Film) . The result indicates that wild VRK1 proteinphosphorylates casein and GST-IkBα (FIG. 13). In contrast, nophosphorylation was observed in reactions with mutant VRK1 carryingmutation in the predicted ATP binding site. This indicates that VRK1 isa protein kinase that contains a typical catalytic domain. In addition,GST protein was not phosphorylated by VRK1, suggesting that thephosphorylation of GST-IkBα protein by VRK1 occurs within IkBα proteinbut not in GST moiety.

[0092] IkBα is said to negatively regulate the function of transcriptionfactor NF-Kb by forming a complex with it. In addition, it is widelyaccepted that IkBα is inactivated by self-phosphorylation, immediatelythereafter undergoes proteolysis, thereby liberating and activatingNF-Kb. NF-Kb is supposed to be a central transcriptional regulator thatinduces a broad range of immune reactions and inflammatory reactions.Therefore, a kinase that phosphorylates IkBα is important as a targetmolecule of anti-inflammatory drugs. Since VRK1 strongly phosphorylatesIkBα in vitro, VRK1 probably participates in the activation of NF-Kb byphosphorylating IkBα in vivo as well. Therefore, it is possible toanticipate anti-inflammatory effects or immunosuppressive effects byinhibiting VRK1 kinase activity, or by reducing its protein amount.

[0093] Next, the requirement for divalent cations in phosphorylation byVRK1 was examined (FIG. 14). In the presence of various divalent cations(Mg, Mn, Zn, and Ca) at a final concentration of 2 Mm or 10 Mm, kinasereactions were performed using casein as a substrate protein. The resultshowed that VRK1 exhibits phosphorylation activity in the presence ofall divalent cations except for Zn. However, the levels of activity weredifferent; VRK1 exhibited especially strong activity in the presence ofMn.

EXAMPLE 10

[0094] Preparation of an Antibody Against VRK1 Protein

[0095] A peptide (SEQ ID NO: 37) corresponding to the C-terminalsequence of the deduced VRK1 amino acid sequence was synthesized (SawadyTechnology) and conjugated to Keyhole limpet hemocyanin (KLH) at itsamino-terminal cysteine mediated bym-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). This was used asan antigen to immunize rabbits, and antisera were obtained. Antibodiesthat specifically react with the peptide were purified from the antiseraby affinity chromatography using Cellulofine (Seikagaku Corporation)conjugated with the peptide. Western blot analysis using extracts ofK562 cells, which were confirmed by northern blot analysis to stronglyexpress VRK1, detected a single band with a molecular weight ofapproximately 50 Kda, indicating that VRK1 protein is specificallyrecognized by the antibody (FIG. 15).

[0096] Industrial Applicability

[0097] The serine-threonine kinase genes isolated by the presentinventors show significant homology to a vaccinia virus gene that isthought to be involved in DNA replication and are strongly expressed inactively growing cells. Furthermore, overexpression of proteins encodedby the genes remarkably enhances cell proliferation activity. Thus, theisolated serine-threonine kinase genes are assumed to participate in theregulation of cell growth. Therefore, it is possible to develop cellgrowth inhibitors or antitumor agents based on a novel mechanism byscreening drugs targeted on the genes (such as antisense DNA), or drugswhich can regulate either the expression of the genes or the activity ofthe proteins encoded by the genes of the present invention.

1 48 1 1662 DNA Homo sapiens CDS (76)...(1263) 1 ccgagttacg agtcggcgaaagcggcggga agttcgtact gggcagaacg cgacgggtct 60 gcggcttagg tgaaa atg cctcgt gta aaa gca gct caa gct gga aga cag 111 Met Pro Arg Val Lys Ala AlaGln Ala Gly Arg Gln 1 5 10 agc tct gca aag aga cat ctt gca gaa caa tttgca gtt gga gag ata 159 Ser Ser Ala Lys Arg His Leu Ala Glu Gln Phe AlaVal Gly Glu Ile 15 20 25 ata act gac atg gca aaa aag gaa tgg aaa gta ggatta ccc att ggc 207 Ile Thr Asp Met Ala Lys Lys Glu Trp Lys Val Gly LeuPro Ile Gly 30 35 40 caa gga ggc ttt ggc tgt ata tat ctt gct gat atg aattct tca gag 255 Gln Gly Gly Phe Gly Cys Ile Tyr Leu Ala Asp Met Asn SerSer Glu 45 50 55 60 tca gtt ggc agt gat gca cct tgt gtt gta aaa gtg gaaccc agt gac 303 Ser Val Gly Ser Asp Ala Pro Cys Val Val Lys Val Glu ProSer Asp 65 70 75 aat gga cct ctt ttt act gaa tta aag ttc tac caa cga gctgca aaa 351 Asn Gly Pro Leu Phe Thr Glu Leu Lys Phe Tyr Gln Arg Ala AlaLys 80 85 90 cca gag caa att cag aaa tgg att cgt acc cgt aag ctg aag tacctg 399 Pro Glu Gln Ile Gln Lys Trp Ile Arg Thr Arg Lys Leu Lys Tyr Leu95 100 105 ggt gtt cct aag tat tgg ggg tct ggt cta cat gac aaa aat ggaaaa 447 Gly Val Pro Lys Tyr Trp Gly Ser Gly Leu His Asp Lys Asn Gly Lys110 115 120 agt tac agg ttt atg ata atg gat cgc ttt ggg agt gac ctt cagaaa 495 Ser Tyr Arg Phe Met Ile Met Asp Arg Phe Gly Ser Asp Leu Gln Lys125 130 135 140 ata tat gaa gca aat gcc aaa agg ttt tct cgg aaa act gtcttg cag 543 Ile Tyr Glu Ala Asn Ala Lys Arg Phe Ser Arg Lys Thr Val LeuGln 145 150 155 cta agc tta aga att ctg gat att ctg gaa tat att cac gagcat gag 591 Leu Ser Leu Arg Ile Leu Asp Ile Leu Glu Tyr Ile His Glu HisGlu 160 165 170 tat gtg cat gga gat atc aag gcc tca aat ctt ctt ctg aactac aag 639 Tyr Val His Gly Asp Ile Lys Ala Ser Asn Leu Leu Leu Asn TyrLys 175 180 185 aat cct gac cag gtg tac ttg gta gat tat ggc ctt gct tatcgg tac 687 Asn Pro Asp Gln Val Tyr Leu Val Asp Tyr Gly Leu Ala Tyr ArgTyr 190 195 200 tgc cca gaa gga gtt cat aaa gaa tac aaa gaa gac ccc aaaaga tgt 735 Cys Pro Glu Gly Val His Lys Glu Tyr Lys Glu Asp Pro Lys ArgCys 205 210 215 220 cac gat ggc act att gaa ttc acg agc atc gat gca cacaat ggt gtg 783 His Asp Gly Thr Ile Glu Phe Thr Ser Ile Asp Ala His AsnGly Val 225 230 235 gcc cca tca aga cgt ggt gat ttg gaa ata ctt ggt tattgc atg atc 831 Ala Pro Ser Arg Arg Gly Asp Leu Glu Ile Leu Gly Tyr CysMet Ile 240 245 250 caa tgg ctt act ggc cat ctt cct tgg gag gat aat ttgaaa gat cct 879 Gln Trp Leu Thr Gly His Leu Pro Trp Glu Asp Asn Leu LysAsp Pro 255 260 265 aaa tat gtt aga gat tcc aaa att aga tac aga gaa aatatt gca agt 927 Lys Tyr Val Arg Asp Ser Lys Ile Arg Tyr Arg Glu Asn IleAla Ser 270 275 280 ttg atg gac aaa tgt ttt cct gag aaa aac aaa cca ggtgaa att gcc 975 Leu Met Asp Lys Cys Phe Pro Glu Lys Asn Lys Pro Gly GluIle Ala 285 290 295 300 aaa tac atg gaa aca gtg aaa tta cta gac tac actgaa aaa cct ctt 1023 Lys Tyr Met Glu Thr Val Lys Leu Leu Asp Tyr Thr GluLys Pro Leu 305 310 315 tat gaa aat tta cgt gac att ctt ttg caa gga ctaaaa gct ata gga 1071 Tyr Glu Asn Leu Arg Asp Ile Leu Leu Gln Gly Leu LysAla Ile Gly 320 325 330 agt aag gat gat ggc aaa ttg gac ctc agt gtt gtggag aat gga ggt 1119 Ser Lys Asp Asp Gly Lys Leu Asp Leu Ser Val Val GluAsn Gly Gly 335 340 345 ttg aaa gca aaa aca ata aca aag aag cga aag aaagaa att gaa gaa 1167 Leu Lys Ala Lys Thr Ile Thr Lys Lys Arg Lys Lys GluIle Glu Glu 350 355 360 agc aag gaa cct ggt gtt gaa gat acg gaa tgg tcaaac aca cag aca 1215 Ser Lys Glu Pro Gly Val Glu Asp Thr Glu Trp Ser AsnThr Gln Thr 365 370 375 380 gag gag gcc ata cag acc cgt tca aga acc agaaag aga gtc cag aag 1263 Glu Glu Ala Ile Gln Thr Arg Ser Arg Thr Arg LysArg Val Gln Lys 385 390 395 taattcagat gctgtgaacc agatttcctt ttctttgttttcttttgact tttttctcct 1323 tttctgttag aactgtttta ttttcctgtg agtcttgcgaggtggaatta atgattaaat 1383 actcatgtgt tcagaaaaca taaacttttt ttataaaaatattttgtaca attcattaaa 1443 ggctaattta tgaaatttga aaatcttcag gttatactccttaagttatc ccaaagccgt 1503 gtgtttgtga tgttttggag tacatatata tgaaaattattatgacacgc acttttctaa 1563 tcattgtaca tttctcagag tggataaaaa tgtttgacaaagtcctcact tttaaggaaa 1623 tgcaaagctt aaaataaaac tctcttttgt ttgatgcag1662 2 396 PRT Homo sapiens 2 Met Pro Arg Val Lys Ala Ala Gln Ala GlyArg Gln Ser Ser Ala Lys 1 5 10 15 Arg His Leu Ala Glu Gln Phe Ala ValGly Glu Ile Ile Thr Asp Met 20 25 30 Ala Lys Lys Glu Trp Lys Val Gly LeuPro Ile Gly Gln Gly Gly Phe 35 40 45 Gly Cys Ile Tyr Leu Ala Asp Met AsnSer Ser Glu Ser Val Gly Ser 50 55 60 Asp Ala Pro Cys Val Val Lys Val GluPro Ser Asp Asn Gly Pro Leu 65 70 75 80 Phe Thr Glu Leu Lys Phe Tyr GlnArg Ala Ala Lys Pro Glu Gln Ile 85 90 95 Gln Lys Trp Ile Arg Thr Arg LysLeu Lys Tyr Leu Gly Val Pro Lys 100 105 110 Tyr Trp Gly Ser Gly Leu HisAsp Lys Asn Gly Lys Ser Tyr Arg Phe 115 120 125 Met Ile Met Asp Arg PheGly Ser Asp Leu Gln Lys Ile Tyr Glu Ala 130 135 140 Asn Ala Lys Arg PheSer Arg Lys Thr Val Leu Gln Leu Ser Leu Arg 145 150 155 160 Ile Leu AspIle Leu Glu Tyr Ile His Glu His Glu Tyr Val His Gly 165 170 175 Asp IleLys Ala Ser Asn Leu Leu Leu Asn Tyr Lys Asn Pro Asp Gln 180 185 190 ValTyr Leu Val Asp Tyr Gly Leu Ala Tyr Arg Tyr Cys Pro Glu Gly 195 200 205Val His Lys Glu Tyr Lys Glu Asp Pro Lys Arg Cys His Asp Gly Thr 210 215220 Ile Glu Phe Thr Ser Ile Asp Ala His Asn Gly Val Ala Pro Ser Arg 225230 235 240 Arg Gly Asp Leu Glu Ile Leu Gly Tyr Cys Met Ile Gln Trp LeuThr 245 250 255 Gly His Leu Pro Trp Glu Asp Asn Leu Lys Asp Pro Lys TyrVal Arg 260 265 270 Asp Ser Lys Ile Arg Tyr Arg Glu Asn Ile Ala Ser LeuMet Asp Lys 275 280 285 Cys Phe Pro Glu Lys Asn Lys Pro Gly Glu Ile AlaLys Tyr Met Glu 290 295 300 Thr Val Lys Leu Leu Asp Tyr Thr Glu Lys ProLeu Tyr Glu Asn Leu 305 310 315 320 Arg Asp Ile Leu Leu Gln Gly Leu LysAla Ile Gly Ser Lys Asp Asp 325 330 335 Gly Lys Leu Asp Leu Ser Val ValGlu Asn Gly Gly Leu Lys Ala Lys 340 345 350 Thr Ile Thr Lys Lys Arg LysLys Glu Ile Glu Glu Ser Lys Glu Pro 355 360 365 Gly Val Glu Asp Thr GluTrp Ser Asn Thr Gln Thr Glu Glu Ala Ile 370 375 380 Gln Thr Arg Ser ArgThr Arg Lys Arg Val Gln Lys 385 390 395 3 1833 DNA Homo sapiens CDS(131)...(1654) 3 ctgcactgcg aggccgacgc agctggagag aagttaggca ggtcctagggagggcaggct 60 cgagtgctgg gcccgcctcc ccgcgggact gtaggcccgg gggctccgcctcgtcgcagc 120 ggcagaagtg atg cca cca aaa aga aat gaa aaa tac aaa cttcct att 169 Met Pro Pro Lys Arg Asn Glu Lys Tyr Lys Leu Pro Ile 1 5 10cca ttt cca gaa ggc aag gtt ctg gat gat atg gaa ggc aat cag tgg 217 ProPhe Pro Glu Gly Lys Val Leu Asp Asp Met Glu Gly Asn Gln Trp 15 20 25 gtactg ggc aag aag att ggc tct gga gga ttt gga ttg ata tat tta 265 Val LeuGly Lys Lys Ile Gly Ser Gly Gly Phe Gly Leu Ile Tyr Leu 30 35 40 45 gctttc ccc aca aat aaa cca gag aaa gat gca aga cat gta gta aaa 313 Ala PhePro Thr Asn Lys Pro Glu Lys Asp Ala Arg His Val Val Lys 50 55 60 gtg gaatat caa gaa aat ggc ccg tta ttt tca gaa ctt aaa ttt tat 361 Val Glu TyrGln Glu Asn Gly Pro Leu Phe Ser Glu Leu Lys Phe Tyr 65 70 75 cag aga gttgca aaa aaa gac tgt atc aaa aag tgg ata gaa cgc aaa 409 Gln Arg Val AlaLys Lys Asp Cys Ile Lys Lys Trp Ile Glu Arg Lys 80 85 90 caa ctt gat tattta gga att cct ctg ttt tat gga tct ggt ctg act 457 Gln Leu Asp Tyr LeuGly Ile Pro Leu Phe Tyr Gly Ser Gly Leu Thr 95 100 105 gaa ttc aag ggaaga agt tac aga ttt atg gta atg gaa aga cta gga 505 Glu Phe Lys Gly ArgSer Tyr Arg Phe Met Val Met Glu Arg Leu Gly 110 115 120 125 ata gat ttacag aag atc tca ggc cag aat ggt acc ttt aaa aag tca 553 Ile Asp Leu GlnLys Ile Ser Gly Gln Asn Gly Thr Phe Lys Lys Ser 130 135 140 act gtc ctgcaa tta ggt atc cga atg ttg gat gta ctg gaa tat ata 601 Thr Val Leu GlnLeu Gly Ile Arg Met Leu Asp Val Leu Glu Tyr Ile 145 150 155 cat gaa aatgaa tat gtt cat ggt gat gta aaa gca gca aat cta ctt 649 His Glu Asn GluTyr Val His Gly Asp Val Lys Ala Ala Asn Leu Leu 160 165 170 ttg ggt tacaaa aat cca gac cag gtt tat ctt gca gat tat gga ctt 697 Leu Gly Tyr LysAsn Pro Asp Gln Val Tyr Leu Ala Asp Tyr Gly Leu 175 180 185 tcc tac agatat tgt ccc aat ggg aac cac aaa cag tat cag gaa aat 745 Ser Tyr Arg TyrCys Pro Asn Gly Asn His Lys Gln Tyr Gln Glu Asn 190 195 200 205 cct agaaaa ggc cat aat ggg aca ata gag ttt acc agc ttg gat gcc 793 Pro Arg LysGly His Asn Gly Thr Ile Glu Phe Thr Ser Leu Asp Ala 210 215 220 cac aaggga gta gcc ttg tcc aga cga agt gac gtt gag atc ctc ggc 841 His Lys GlyVal Ala Leu Ser Arg Arg Ser Asp Val Glu Ile Leu Gly 225 230 235 tac tgcatg ctg cgg tgg ttg tgt ggg aaa ctt ccc tgg gaa cag aac 889 Tyr Cys MetLeu Arg Trp Leu Cys Gly Lys Leu Pro Trp Glu Gln Asn 240 245 250 ctg aaggac cct gtg gct gtg cag act gct aaa aca aat ctg ttg gac 937 Leu Lys AspPro Val Ala Val Gln Thr Ala Lys Thr Asn Leu Leu Asp 255 260 265 gag ctcccc cag tca gtg ctt aaa tgg gct cct tct gga agc agt tgc 985 Glu Leu ProGln Ser Val Leu Lys Trp Ala Pro Ser Gly Ser Ser Cys 270 275 280 285 tgtgaa ata gcc caa ttt ttg gta tgt gct cat agt tta gca tat gat 1033 Cys GluIle Ala Gln Phe Leu Val Cys Ala His Ser Leu Ala Tyr Asp 290 295 300 gaaaag cca aac tat caa gcc ctc aag aaa att ttg aac cct cat gga 1081 Glu LysPro Asn Tyr Gln Ala Leu Lys Lys Ile Leu Asn Pro His Gly 305 310 315 atacct tta gga cca ctg gac ttt tcc aca aaa gga cag agt ata aat 1129 Ile ProLeu Gly Pro Leu Asp Phe Ser Thr Lys Gly Gln Ser Ile Asn 320 325 330 gtccat act cca aac agt caa aaa gtt gat tca caa aag gct gca aca 1177 Val HisThr Pro Asn Ser Gln Lys Val Asp Ser Gln Lys Ala Ala Thr 335 340 345 aagcaa gtc aac aag gca cac aat agg tta atc gaa aaa aaa gtc cac 1225 Lys GlnVal Asn Lys Ala His Asn Arg Leu Ile Glu Lys Lys Val His 350 355 360 365agt gag aga agc gct gag tcc tgt gca aca tgg aaa gtg cag aaa gag 1273 SerGlu Arg Ser Ala Glu Ser Cys Ala Thr Trp Lys Val Gln Lys Glu 370 375 380gag aaa ctg att gga ttg atg aac aat gaa gca gct cag gaa agc aca 1321 GluLys Leu Ile Gly Leu Met Asn Asn Glu Ala Ala Gln Glu Ser Thr 385 390 395agg aga aga cag aaa tat caa gag tct caa gaa cct ttg aat gaa gta 1369 ArgArg Arg Gln Lys Tyr Gln Glu Ser Gln Glu Pro Leu Asn Glu Val 400 405 410aac agt ttc cca caa aaa atc agc tat aca caa ttc cca aac tca ttt 1417 AsnSer Phe Pro Gln Lys Ile Ser Tyr Thr Gln Phe Pro Asn Ser Phe 415 420 425tat gag cct cat caa gat ttt acc agt cca gat ata ttc aag aag tca 1465 TyrGlu Pro His Gln Asp Phe Thr Ser Pro Asp Ile Phe Lys Lys Ser 430 435 440445 aga tct cca tct tgg tat aaa tac act tcc aca gtc agc acg ggg atc 1513Arg Ser Pro Ser Trp Tyr Lys Tyr Thr Ser Thr Val Ser Thr Gly Ile 450 455460 aca gac tta gaa agt tca act gga ctt tgg cct aca att tcc cag ttt 1561Thr Asp Leu Glu Ser Ser Thr Gly Leu Trp Pro Thr Ile Ser Gln Phe 465 470475 act ctt agt gaa gag aca aac gca gat gtt tat tat tat cgc atc atc 1609Thr Leu Ser Glu Glu Thr Asn Ala Asp Val Tyr Tyr Tyr Arg Ile Ile 480 485490 ata cct gtc ctt ttg atg tta gta ttt ctt gct tta ttt ttt ctc 1654 IlePro Val Leu Leu Met Leu Val Phe Leu Ala Leu Phe Phe Leu 495 500 505tgaagatgat accaaaattc cttttgataa ttttttaagt ttccagctct tcaccgaaat 1714gttgtattct tatttcagtg tttccttcca gacattttta aggtaattgg ctttaaaaag 1774agaacatatt ttaacaaagt ttgtggacac tctaaaaaat aaaattgctt tgtactagt 1833 4508 PRT Homo sapiens 4 Met Pro Pro Lys Arg Asn Glu Lys Tyr Lys Leu ProIle Pro Phe Pro 1 5 10 15 Glu Gly Lys Val Leu Asp Asp Met Glu Gly AsnGln Trp Val Leu Gly 20 25 30 Lys Lys Ile Gly Ser Gly Gly Phe Gly Leu IleTyr Leu Ala Phe Pro 35 40 45 Thr Asn Lys Pro Glu Lys Asp Ala Arg His ValVal Lys Val Glu Tyr 50 55 60 Gln Glu Asn Gly Pro Leu Phe Ser Glu Leu LysPhe Tyr Gln Arg Val 65 70 75 80 Ala Lys Lys Asp Cys Ile Lys Lys Trp IleGlu Arg Lys Gln Leu Asp 85 90 95 Tyr Leu Gly Ile Pro Leu Phe Tyr Gly SerGly Leu Thr Glu Phe Lys 100 105 110 Gly Arg Ser Tyr Arg Phe Met Val MetGlu Arg Leu Gly Ile Asp Leu 115 120 125 Gln Lys Ile Ser Gly Gln Asn GlyThr Phe Lys Lys Ser Thr Val Leu 130 135 140 Gln Leu Gly Ile Arg Met LeuAsp Val Leu Glu Tyr Ile His Glu Asn 145 150 155 160 Glu Tyr Val His GlyAsp Val Lys Ala Ala Asn Leu Leu Leu Gly Tyr 165 170 175 Lys Asn Pro AspGln Val Tyr Leu Ala Asp Tyr Gly Leu Ser Tyr Arg 180 185 190 Tyr Cys ProAsn Gly Asn His Lys Gln Tyr Gln Glu Asn Pro Arg Lys 195 200 205 Gly HisAsn Gly Thr Ile Glu Phe Thr Ser Leu Asp Ala His Lys Gly 210 215 220 ValAla Leu Ser Arg Arg Ser Asp Val Glu Ile Leu Gly Tyr Cys Met 225 230 235240 Leu Arg Trp Leu Cys Gly Lys Leu Pro Trp Glu Gln Asn Leu Lys Asp 245250 255 Pro Val Ala Val Gln Thr Ala Lys Thr Asn Leu Leu Asp Glu Leu Pro260 265 270 Gln Ser Val Leu Lys Trp Ala Pro Ser Gly Ser Ser Cys Cys GluIle 275 280 285 Ala Gln Phe Leu Val Cys Ala His Ser Leu Ala Tyr Asp GluLys Pro 290 295 300 Asn Tyr Gln Ala Leu Lys Lys Ile Leu Asn Pro His GlyIle Pro Leu 305 310 315 320 Gly Pro Leu Asp Phe Ser Thr Lys Gly Gln SerIle Asn Val His Thr 325 330 335 Pro Asn Ser Gln Lys Val Asp Ser Gln LysAla Ala Thr Lys Gln Val 340 345 350 Asn Lys Ala His Asn Arg Leu Ile GluLys Lys Val His Ser Glu Arg 355 360 365 Ser Ala Glu Ser Cys Ala Thr TrpLys Val Gln Lys Glu Glu Lys Leu 370 375 380 Ile Gly Leu Met Asn Asn GluAla Ala Gln Glu Ser Thr Arg Arg Arg 385 390 395 400 Gln Lys Tyr Gln GluSer Gln Glu Pro Leu Asn Glu Val Asn Ser Phe 405 410 415 Pro Gln Lys IleSer Tyr Thr Gln Phe Pro Asn Ser Phe Tyr Glu Pro 420 425 430 His Gln AspPhe Thr Ser Pro Asp Ile Phe Lys Lys Ser Arg Ser Pro 435 440 445 Ser TrpTyr Lys Tyr Thr Ser Thr Val Ser Thr Gly Ile Thr Asp Leu 450 455 460 GluSer Ser Thr Gly Leu Trp Pro Thr Ile Ser Gln Phe Thr Leu Ser 465 470 475480 Glu Glu Thr Asn Ala Asp Val Tyr Tyr Tyr Arg Ile Ile Ile Pro Val 485490 495 Leu Leu Met Leu Val Phe Leu Ala Leu Phe Phe Leu 500 505 5 22 DNAArtificial Sequence synthetically generated primer 5 ctaatacgactcactatagg gc 22 6 21 DNA Artificial Sequence synthetically generatedprimer 6 tgtagcgtga agacgacaga a 21 7 22 DNA Artificial Sequencesynthetically generated primer 7 tcgagcggcc gcccgggcag gt 22 8 22 DNAArtificial Sequence synthetically generated primer 8 agggcgtggtgcggagggcg gt 22 9 22 DNA Artificial Sequence synthetically generatedprimer 9 ccagggtttt cccagtcacg ac 22 10 22 DNA Artificial Sequencesynthetically generated primer 10 tcacacagga aacagctatg ac 22 11 24 DNAArtificial Sequence synthetically generated primer 11 tgtagttcagaagaagattt gagg 24 12 24 DNA Artificial Sequence synthetically generatedprimer 12 ataatggatc gctttgggag tgac 24 13 26 DNA Artificial Sequencesynthetically generated primer 13 tgaaggtcgg agtcaacgga tttggt 26 14 24DNA Artificial Sequence synthetically generated primer 14 catgtgggccatgaggtcca ccac 24 15 27 DNA Artificial Sequence synthetically generatedprimer 15 ccatcctaat acgactcact atagggc 27 16 24 DNA Artificial Sequencesynthetically generated primer 16 ggattttcct gatactgttt gtgg 24 17 24DNA Artificial Sequence synthetically generated primer 17 accacaaacagtatcaggaa aatc 24 18 24 DNA Artificial Sequence synthetically generatedprimer 18 acctttaaaa agtcaactgt cctg 24 19 24 DNA Artificial Sequencesynthetically generated primer 19 aaaaattatc aaaaggaatt ttgg 24 20 25DNA Artificial Sequence synthetically generated primer 20 ttactcttagtgaagagaca aacgc 25 21 36 DNA Artificial Sequence syntheticallygenerated primer 21 agctgcggcc gcggtctgcg gcttaggtga aaatgc 36 22 36 DNAArtificial Sequence synthetically generated primer 22 agctgcggccgcaaaacaaa gaaaaggaaa tctggt 36 23 37 DNA Artificial Sequencesynthetically generated primer 23 agctgcggcc gcaagtgatg ccaccaaaaagaaatga 37 24 36 DNA Artificial Sequence synthetically generated primer24 agctgcggcc gctggaagga aacactgaaa taagaa 36 25 10 PRT ArtificialSequence synthetically generated peptide 25 Glu Gln Lys Leu Ile Ser GluGlu Asp Leu 1 5 10 26 33 DNA Artificial Sequence synthetically generatedprimer 26 gatggatccg gtctgcggct taggtgaaaa tgc 33 27 60 DNA ArtificialSequence synthetically generated primer 27 gatggatcct tagaggtcttcttctgagat gagcttctgc tccttctgga ctctctttct 60 28 33 DNA ArtificialSequence synthetically generated primer 28 gatggatcca gtgatgccaccaaaaagaaa tga 33 29 60 DNA Artificial Sequence synthetically generatedprimer 29 gatggatcct tagaggtctt cttctgagat gagcttctgc tcgagaaaaaataaagcaag 60 30 31 DNA Artificial Sequence synthetically generatedprimer 30 gatggatccc catgcctcgt gtaaaagcag c 31 31 31 DNA ArtificialSequence synthetically generated primer 31 gatggatccc ccaaagaaaaggaaatctgg t 31 32 31 DNA Artificial Sequence synthetically generatedprimer 32 gatggatccc catgccacca aaaagaaatg a 31 33 31 DNA ArtificialSequence synthetically generated primer 33 gatggatccc cacaacatttcggtgaagag c 31 34 29 DNA Artificial Sequence synthetically generatedprimer 34 ccttgtgttg tatgggtgga acccagtga 29 35 31 DNA ArtificialSequence synthetically generated primer 35 acaccacgat gcctggagcaatggcaacaa c 31 36 25 PRT Homo sapiens 36 Met Leu Pro Glu Ser Glu AspGlu Glu Ser Tyr Asp Thr Glu Ser Glu 1 5 10 15 Phe Thr Glu Phe Thr GluAsp Glu Leu 20 25 37 19 PRT Homo sapiens 37 Cys Gln Thr Glu Glu Ala IleGln Thr Arg Ser Arg Thr Arg Lys Arg 1 5 10 15 Val Gln Lys 38 44 DNAArtificial Sequence synthetically generated oligonucleotide 38ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44 39 10 DNA ArtificialSequence synthetically generated oligonucleotide 39 acctgcccgg 10 40 43DNA Artificial Sequence synthetically generated oligonucleotide 40tgtagcgtga agacgacaga aagggcgtgg tgcggagggc ggt 43 41 11 DNA ArtificialSequence synthetically generated oligonucleotide 41 accgccctcc g 11 42261 DNA Artificial Sequence CDS (1)...(261) synthetically generatedoligonucleotide 42 acc tgg gtg ttc cta agt att ggg ggt ctg gtc tac atgaca aaa atg 48 Thr Trp Val Phe Leu Ser Ile Gly Gly Leu Val Tyr Met ThrLys Met 1 5 10 15 gaa aaa gtt aca ggt tta tga taa tgg atc gct ttg ggagtg acc ttc 96 Glu Lys Val Thr Gly Leu * * Trp Ile Ala Leu Gly Val ThrPhe 20 25 30 aga aaa tat atg aag caa atg cca aaa ggt ttt ctc gga aaa ctgtct 144 Arg Lys Tyr Met Lys Gln Met Pro Lys Gly Phe Leu Gly Lys Leu Ser35 40 45 tgc agc taa gct taa gaa ttc tgg ata ttc tgg aat ata ttc acg agc192 Cys Ser * Ala * Glu Phe Trp Ile Phe Trp Asn Ile Phe Thr Ser 50 55 60atg agt atg tgc atg gag ata tca agg cct caa atc ttc ttc tga act 240 MetSer Met Cys Met Glu Ile Ser Arg Pro Gln Ile Phe Phe * Thr 65 70 75 acaaga atc ctg acc agg tgt 261 Thr Arg Ile Leu Thr Arg Cys 80 43 82 PRTArtificial Sequence synthetically generated peptide 43 Thr Trp Val PheLeu Ser Ile Gly Gly Leu Val Tyr Met Thr Lys Met 1 5 10 15 Glu Lys ValThr Gly Leu Trp Ile Ala Leu Gly Val Thr Phe Arg Lys 20 25 30 Tyr Met LysGln Met Pro Lys Gly Phe Leu Gly Lys Leu Ser Cys Ser 35 40 45 Ala Glu PheTrp Ile Phe Trp Asn Ile Phe Thr Ser Met Ser Met Cys 50 55 60 Met Glu IleSer Arg Pro Gln Ile Phe Phe Thr Thr Arg Ile Leu Thr 65 70 75 80 Arg Cys44 261 DNA Artificial Sequence CDS (2)...(259) synthetically generatedoligonucleotide 44 a cct ggg tgt tcc taa gta ttg ggg gtc tgg tct aca tgacaa aaa tgg 49 Pro Gly Cys Ser * Val Leu Gly Val Trp Ser Thr * Gln LysTrp 1 5 10 aaa aag tta cag gtt tat gat aat gga tcg ctt tgg gag tga ccttca 97 Lys Lys Leu Gln Val Tyr Asp Asn Gly Ser Leu Trp Glu * Pro Ser 1520 25 gaa aat ata tga agc aaa tgc caa aag gtt ttc tcg gaa aac tgt ctt145 Glu Asn Ile * Ser Lys Cys Gln Lys Val Phe Ser Glu Asn Cys Leu 30 3540 gca gct aag ctt aag aat tct gga tat tct gga ata tat tca cga gca 193Ala Ala Lys Leu Lys Asn Ser Gly Tyr Ser Gly Ile Tyr Ser Arg Ala 45 50 5560 tga gta tgt gca tgg aga tat caa ggc ctc aaa tct tct tct gaa cta 241 *Val Cys Ala Trp Arg Tyr Gln Gly Leu Lys Ser Ser Ser Glu Leu 65 70 75 caagaa tcc tga cca ggt gt 261 Gln Glu Ser * Pro Gly 80 45 80 PRT ArtificialSequence synthetically generated peptide 45 Pro Gly Cys Ser Val Leu GlyVal Trp Ser Thr Gln Lys Trp Lys Lys 1 5 10 15 Leu Gln Val Tyr Asp AsnGly Ser Leu Trp Glu Pro Ser Glu Asn Ile 20 25 30 Ser Lys Cys Gln Lys ValPhe Ser Glu Asn Cys Leu Ala Ala Lys Leu 35 40 45 Lys Asn Ser Gly Tyr SerGly Ile Tyr Ser Arg Ala Val Cys Ala Trp 50 55 60 Arg Tyr Gln Gly Leu LysSer Ser Ser Glu Leu Gln Glu Ser Pro Gly 65 70 75 80 46 261 DNAArtificial Sequence CDS (3)...(260) synthetically generatedoligonucleotide 46 ac ctg ggt gtt cct aag tat tgg ggg tct ggt cta catgac aaa aat 47 Leu Gly Val Pro Lys Tyr Trp Gly Ser Gly Leu His Asp LysAsn 1 5 10 15 gga aaa agt tac agg ttt atg ata atg gat cgc ttt ggg agtgac ctt 95 Gly Lys Ser Tyr Arg Phe Met Ile Met Asp Arg Phe Gly Ser AspLeu 20 25 30 cag aaa ata tat gaa gca aat gcc aaa agg ttt tct cgg aaa actgtc 143 Gln Lys Ile Tyr Glu Ala Asn Ala Lys Arg Phe Ser Arg Lys Thr Val35 40 45 ttg cag cta agc tta aga att ctg gat att ctg gaa tat att cac gag191 Leu Gln Leu Ser Leu Arg Ile Leu Asp Ile Leu Glu Tyr Ile His Glu 5055 60 cat gag tat gtg cat gga gat atc aag gcc tca aat ctt ctt ctg aac239 His Glu Tyr Val His Gly Asp Ile Lys Ala Ser Asn Leu Leu Leu Asn 6570 75 tac aag aat cct gac cag gtg t 261 Tyr Lys Asn Pro Asp Gln Val 8085 47 86 PRT Artificial Sequence synthetically generated peptide 47 LeuGly Val Pro Lys Tyr Trp Gly Ser Gly Leu His Asp Lys Asn Gly 1 5 10 15Lys Ser Tyr Arg Phe Met Ile Met Asp Arg Phe Gly Ser Asp Leu Gln 20 25 30Lys Ile Tyr Glu Ala Asn Ala Lys Arg Phe Ser Arg Lys Thr Val Leu 35 40 45Gln Leu Ser Leu Arg Ile Leu Asp Ile Leu Glu Tyr Ile His Glu His 50 55 60Glu Tyr Val His Gly Asp Ile Lys Ala Ser Asn Leu Leu Leu Asn Tyr 65 70 7580 Lys Asn Pro Asp Gln Val 85 48 300 PRT Homo sapiens 48 Met Asn Phe GlnGly Leu Val Leu Thr Asp Asn Cys Lys Asn Gln Trp 1 5 10 15 Val Val GlyPro Leu Ile Gly Lys Gly Gly Phe Gly Ser Ile Tyr Thr 20 25 30 Thr Asn AspAsn Asn Tyr Val Val Lys Ile Glu Pro Lys Ala Asn Gly 35 40 45 Ser Leu PheThr Glu Gln Ala Phe Tyr Thr Arg Val Leu Lys Pro Ser 50 55 60 Val Ile GluGlu Trp Lys Lys Ser His Asn Ile Lys His Val Gly Leu 65 70 75 80 Ile ThrCys Lys Ala Phe Gly Leu Tyr Lys Ser Ile Asn Val Glu Tyr 85 90 95 Arg GluLeu Val Ile Asn Arg Leu Gly Ala Asp Leu Asp Ala Val Ile 100 105 110 ArgAla Asn Asn Asn Arg Leu Pro Lys Arg Ser Val Met Leu Ile Gly 115 120 125Ile Glu Ile Leu Asn Thr Ile Gln Phe Met His Glu Gln Gly Tyr Ser 130 135140 His Gly Asp Ile Lys Ala Ser Asn Ile Val Leu Asp Gln Ile Asp Lys 145150 155 160 Asn Lys Leu Tyr Leu Val Asp Tyr Gly Leu Val Ser Lys Phe MetSer 165 170 175 Asn Gly Glu His Val Pro Phe Ile Arg Asn Pro Asn Lys MetAsp Asn 180 185 190 Gly Thr Ile Glu Phe Thr Pro Ile Asp Ser His Lys GlyTyr Val Val 195 200 205 Ser Arg Arg Gly Asp Leu Glu Thr Leu Gly Tyr CysMet Ile Arg Trp 210 215 220 Leu Gly Gly Ile Leu Pro Trp Thr Lys Ile SerGlu Thr Lys Asn Cys 225 230 235 240 Ala Leu Val Ser Ala Thr Lys Gln LysTyr Val Asn Asn Thr Ala Thr 245 250 255 Leu Leu Met Thr Ser Leu Gln TyrAla Pro Arg Glu Leu Leu Gln Tyr 260 265 270 Ile Thr Met Val Asn Ser LeuThr Tyr Phe Glu Glu Pro Asn Tyr Asp 275 280 285 Glu Phe Arg His Ile LeuMet Gln Gly Val Tyr Tyr 290 295 300

What is claimed is:
 1. A protein having the amino acid sequence of SEQID NO: 2, or a protein having the same amino acid sequence where one ormore amino acids are added, deleted, or substituted and havingserine-threonine kinase activity.
 2. A protein having the amino acidsequence of SEQ ID NO: 4, or a protein having the same amino acidsequence where one or more amino acids are added, deleted, orsubstituted and having serine-threonine kinase activity.
 3. A proteinencoded by a DNA sequence that hybridizes with the DNA sequence of SEQID NO: 1 or its complementary sequence and having serine-threoninekinase activity.
 4. A protein encoded by a DNA sequence that hybridizeswith the DNA sequence of SEQ ID NO: 3 or its complementary sequence andhaving serine-threonine kinase activity.
 5. A DNA encoding the proteinof claim
 1. 6. A vector comprising the DNA of claim
 5. 7. A transformantcarrying the vector of claim
 6. 8. A method of producing a protein,wherein the method comprises cultivating the transformant of claim
 7. 9.An antibody binding to the protein of claim
 1. 10. An antisense DNAagainst the DNA of claim 5 or part of it.
 11. A method of screeningcompounds having inhibitory activity of serine-threonine kinase activityof the protein of claim 1, wherein the method comprises (a) contactingthe protein of claim 1 with a substrate to be phosphorylated by thisprotein in the presence of a test compound to detect the kinase activityof the protein of claim 1, and (b) comparing the kinase activitydetected in step (a) with that detected in the absence of the testcompound and selecting a compound that lowers the kinase activity of theprotein of claim 1.